Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Blood clot formation occurs through the conversion of fibrinogen by thrombin and Factor XIIIa to form a cross-linked fibrin polymer. Fibrinogen is a 340,000-Da dimeric glycoprotein composed of six disulphide-linked polypeptide chains: two Aα(Mr=65,000), two Bβ(Mr=56,000), and two γ(Mr=47,000). Fibrinogen is converted to fibrin through limited proteolysis by thrombin, which exposes polymerization sites in fibrinogen (Kudryk et al. (1974) J. Biol. Chem. 249:3322-3325). The fibrin monomers spontaneously associate with each other to form the web-like fibrin clot (Blombäck (1996) Thromb. Res. 83:1-75).
Factor XIIIa is a plasma transglutaminase that strengthens the fibrin clot by forming covalent bonds between adjacent fibrin monomers (Lorand et al. (1993) Methods Enzymol. 222:22-35). Plasma Factor XIII is a 320,000-Da tetrameric protein composed of two polypeptide a chains (Mr=83,000) and two polypeptide b chains (Mr=80,000; Schwartz et al. (1973) J. Biol. Chem. 248:1395-1407). Factor XIII normally circulates as an inactive proenzyme until it is activated by thrombin cleavage of a 4000-Da activation peptide from each a subunit, which is followed by the dissociation of the b subunits. Activated factor XIII, or XIIIa, catalyzes the formation of γ-glutamyl-ε-lysine bonds between polypeptide chains in fibrin (Chen et al. (1969) Proc. Natl. Acad. Sci. U.S.A. 63:420-427). These cross-links strengthen the fibrin clot (Lorand (1972) Ann. N. Y. Acad. Sci. 202:6-30) and increase its resistance to lysis (Gaffney and Whitaker (1979) Thromb. Res. 14:85-94; Reed et al. (1992) Thromb. Haemostasis 68:315-320; Siebenlist and Mosesson (1994) J. Biol. Chem. 269:28414-28419).
Trauma is the leading cause of death for people between the ages of 1 and 44 in the United States (Bonne et al., eds. “Reducing the Burden of Injury: Advancing Prevention and Treatment.” Committee on Injury Prevention and Control, Institute of Medicine (Washington, D.C., National Academy Press, 1999). The majority of deaths that occur during the first 48 hours following a traumatic event are the result of uncontrolled bleeding (Sauaia et al. (1995) “Epidemiology of Trauma Deaths: A Reassessment” J. Trauma 38:185-193). A common result of traumatic injury is disseminated intravascular coagulation (DIC), in which the activation of fibrinolytic enzymes causes the clot to dissolve. Massive hemorrhage can be resistant even to high doses of recombinant factor VIIa. The primary treatment of such injuries is therefore surgical repair, which is often aided by the use of fibrin sealants to stop hemorrhage. Fibrin sealants, such as BERIPLAST-P™ (Aventis-Behring), CROSSEAL™ (Johnson & Johnson), and TISSEEL™ (Baxter) may be applied during surgery from a two-syringe system. One syringe contains the fibrin precursor protein, fibrinogen, and the other syringe contains the clotting factor thrombin. These two components may be forced into a mixing chamber and act much like a two-part epoxy resin in which fibrinogen serves as the resin and thrombin serves as the catalyst. The mixture coagulates within minutes and stops bleeding from the wound site.
Fibrinolytic enzymes that are activated in DIC can, however, digest the applied fibrin sealant, resulting in re-bleeding of the wound even after initial control of hemorrhage. Furthermore, inhibitors of the fibrinolytic enzymes that are sometimes added to fibrin sealant, such as aprotinin, can be immunogenic and cause anaphylactic reactions. Therefore, there is still a need for degradation resistant fibrin sealants which avoid these drawbacks.